Akira Tokunaga

Dendritic patterns of neurons in the rat superior colliculus

Abstract The cell type of the rat superior colliculus was investigated using the modified Golgi-Cox method. The superior colliculus was found to be composed of two laminar sets, which develop different neuronal organizations. The superficial layers, containing the zonal-to-optic strata, consisted of many neurons of variable size and shape. However, on the basis of the profile and the orientation of the dendritic fields, cells of the superficial layers were classified into four types: the horizontal, the cylindrical, the reversed conical, and the multipolar cells. Some of the cells were further subdivided according to the orientation and the width of dendritic fields. The deep layers which included the middle gray to deep white laminae were characterized by medium-sized and large multipolar neurons. In addition to these cells with global or ellipsoidal dendritic fields, another two types of cells, vertical and horizontal neurons, were also included.

The organization of neurons in the nucleus of the lateral lemniscus projecting to the superior and inferior colliculi in the rat

The topographic organization of neurons in the dorsal nucleus of the lateral lemniscus (DNLL) which project to the superior and inferior colliculi was studied using the retrograde horseradish peroxidase (HRP) and the fluorescent double labeling methods. Neurons projecting to the superior colliculus (SC) are situated in the rostral portion of the DNLL, whereas those to the inferior colliculus (IC) are found in the caudal area of this nucleus. These two portions are completely separated from each other and no neurons projecting to both the SC and the IC are observed. In the dorsolateral part of the rostral portion of the DNLL, neurons projecting to the ipsilateral SC are found, whereas neurons projecting to the contralateral SC are located in the central to medial part of the nucleus, but no neurons sending collateral axons to both sides of the SC were observed. Neurons located in the central part of the caudal area of the DNLL project to the ipsilateral IC and neurons in the lateral and medial parts project contralaterally to the IC. Some of the neurons in the caudal part of the DNLL have divergent axonal branching projecting to both sides of the IC. In the ventral nucleus of the lateral lemniscus, labeled neurons were observed only when the HRP was injected into the ipsilateral IC.

Immunohistochemical analyses of DNA topoisomerase II isoforms in developing rat cerebellum.

In mammalian cells, two isoforms of DNA topoisomerase II (topo IIα and topo IIβ) have been identified. Topo IIα is essential in mitotic cells, whereas the function of topo IIβ remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform‐specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo IIα immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo IIβ immunoreactivity. The postmitotic granule cells in the postnatal EGL showed an abrupt transition of expressed topo II isoforms from IIα to IIβ. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo IIβ immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo IIβ signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplasmic signal decreased significantly as the cells reached terminal differentiation. The residual topo IIβ in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo IIβ localized in nucleoplasm is the functional entity involved in neuronal differentiation. J. Comp. Neurol. 431:228–239, 2001.

Laminar origin of the tecto-thalamic projections in the albino rat.

Cells of origin of the tecto-LP (lateroposterior nucleus of the thalamus) projection and the tecto-LGNd (dorsal nucleus of the lateral geniculate body) projection were studied in the albino rat by means of retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Tecto-LGNd neurons with small spindle form were located in the stratum griseum superficiale of the superior colliculus (SC), whereas tecto-LP neurons with polygonal shape were found in the stratum opticum of the SC.

Neuronal organization of the corpus parabigeminum in the rat

Abstract The cytoarchitecture and the cell type of the rat parabigeminal body were studied. The parabigeminal nucleus was divided into three small subgroups. The middle subgroup was found to be packed mainly with 10- to 16-μm rounded or polygonal cells, which were stained deeply. Fusiform cells of 10 to 16 × 6 to 8 μm formed the dorsal and ventral portions. The parabigeminal neurons which were impregnated with the modified Golgi-Cox method could be classified into four cell types, according to the whole profile, orientation, and width of the dendritic field. Neurons with the pyramidal dendritic field were found in the middle subgroup of the structure and the axons emerged medially toward the mesencephalic reticular formation. They were considered to be projection cells. Nerve cells with the hemispherical dendritic field were common in the dorsal and ventral parabigeminal subgroups. The arrangement of the dendritic field of the aforementioned neurons was variable from cell to cell, reflecting differences in their plane of sectioning. The cylindrical neurons appeared in the medial part of the dorsal parabigeminal subgroup. Their dendrites extended along the medial and dorsal boundary of the nucleus. Both the hemispherical and cylindrical cells were judged to be intrinsic neurons because no axon exceeded beyond the whole extent of the parabigeminal body. There is a probability that the multipolar neurons are aberrant reticular elements.

Primary and secondary subcortical projections of the monkey visual system. An autoradiographic study

Recent studies on the primary visual pathways carried out by means of autoradiographic tracer techniques15,1~,20, 21, have considerably improved our knowledge on the terminations of retinal fibers in diencephalic and mesencephalic areas. In addition, transsynaptic transport to secondary retinal target sites has also been observed in a number of speciesS,9, 22. Since pertinent data in the monkey are still scarce, we decided to study transsynaptic transport along the visual projection pathways in this animal and to pay special attention to pretectal and mesencephalic areas. The results are briefly summarized in this report. An equal mixture of [3H]fucose and [aH]proline, 2-6 mCi in 50-100/~I was injected in the vitreous of one eye in 5 monkeys (2 adult Macaca fascieularis and 3 young Erythroeebus patas). One macaque (A79-188) received a second injection 7 days after the first injection, 12 mCi in total. The animals were allowed to survive from 11 to 21 days after the first injection and then were perfused with buffered 4 ~ paraformaldehyde. The brains were cut in either frozen or paraffin sections, and processed for autoradiography, exposed for several weeks at 4 °C, then developed and counterstained with cresyl violet. The density of silver grains was determined at a magnification of 600 × with an eyepiece reticule consisting of 100 squares (each square ---625 sq./~m) which was placed at random over the nucleus to be analyzed; silver grains in 60-100 squares were counted and the mean grain density per unit area (625 sq.#m) in each nuclear region was determined. The mean grain density over subependymal layers in the lateral wall of the aqueduct was regarded as background level in each case. Statistical differences were examined by the Students t-test.

Filopodia and growth cones in the vertically migrating granule cells of the postnatal mouse cerebellum

Abstract The details of the morphology of vertically migrating granule cells were examined semiquantitatively in the postnatal mouse cerebellum by a Golgi method, with special reference to the growth cone-related structures such as filopodia and lamellipodia. The first sign of inward migration was extension of short, vertical filopodium-like processes from the sides of the perikarya of tangentially oriented granule cells, followed by a change of orientation of cell bodies to the vertical axis showing a T-shaped morphology. The T-shaped migratory cells formed sprouted filopodia (side spikes) from their vertical leading processes and perikarya at right angles to the vertical axis. More than three-quarters of the migratory cells extended the side spikes. The presence of such side spikes was confirmed with laser scanning confocal microscopy of granule cells labeled with 1,1′, dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate and also with transmission electron microscopy (TEM). In addition, about one-fourth of migratory cells extended lamellipodia of web-like forms along the stem or at the tip of the leading process, some of which showed a typical growth cone. Several morphological variations of vertical granule cells were also observed. Furthermore, TEM observation confirmed that side spikes from migratory cells made direct contact with parallel fibers. The present results suggest that, during vertical migration, growth cone-related structures of the leading processes of granule cells adhere to and probably recognize tangentially oriented parallel fibers. Therefore, the mechanisms of the vertical guidance and migration of granule cells in the cerebellar cortex seem to be multiple, involving not only parallel contact guidance by the Bergmann glia fibers but also perpendicular contact guidance by the parallel fibers. These parallel and perpendicular geometric cues surrounding the granule cells seem to have produced the varying morphology of vertically migrating granule cells.

Retinal Projections in the House Musk Shrew, Suncus murinus, as Determined by Anterograde Transport of WGA-HRP

Retinal projections in the house musk shrew (Suncus murinus) were determined by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Unilateral injection of WGA-HRP into the vitreous body resulted in the terminal labeling of the optic projections in the suprachiasmatic nucleus (SCH), the ventral (CGLv) and dorsal (CGLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the pretectum, the superficial layers of the superior colliculus (CS), and the dorsal terminal nucleus (DTN) of the accessory optic system (AOS). Labeling of the SCH was bilateral, with ipsilateral predominance, and covered the whole dorsoventral extent of the nucleus. Immunohistochemical studies revealed that VIP-like immunoreactive neurons and fibers were present in almost all parts of the SCH. No hypothalamic regions other than the SCH received the optic fibers. The ipsilateral projections to the CGLv, CGLd, and IGL were sparse, and a considerable number of uncrossed retinal fibers were found in the pretectal olivary nucleus. No retinal projections to the lateral posterior thalamic nucleus (LP) were found. Ipsilateral optic fibers projected sparsely to the medial part of the CS. The AOS consisted of a small DTN with a very few crossed retinal projections but no lateral and medial terminal nuclei. In addition, the AOS had no inferior fascicle.

Morphology of neurites from N18TG2 cell induced by protein kinase inhibitor H-7 and by cAMP

Protein kinase inhibitor H-7 and dibutyryl (dB)-cAMP were found to induce neuritic processes in mouse neuroblastoma N18TG2 cells (36). In the present study, morphological differences between the neurites induced by H-7 and those by dB-cAMP were examined using electron microscopy (TEM and SEM) and tubulin immunohistochemistry. It was observed that: 1) The neurites induced by H-7 were relatively thin and frequently had varicosities. On the other hand, the neurites induced by dB-cAMP were thick but they had few varicosities. 2) Centrioles were frequently observed in the cells treated with dB-cAMP but were not encountered in the H-7-treated cells. 3) TEM and tubulin immunohistochemistry revealed that the main shafts of the neurites induced either by H-7 or dB-cAMP were filled with microtubules, but that the varicosities induced by H-7 contained a smaller amount of microtubules. 4) The stability to colchicine was greater in the neurites induced by H-7 than in those by dB-cAMP. From these features of the neurites, it was inferred that neurite outgrowth induced by dB-cAMP is deeply related to the formation of microtubules and that the neurites induced by H-7 were involved in other processes probably including an adhesive property of cell surfaces.

The reciprocal connections of the suprageniculate nucleus and the superior colliculus in the rat

Reciprocally bilateral connections between the superior colliculus and the suprageniculate nucleus have been studied in the rat, using the anterograde and retrograde transport techniques of HRP. In those cases where the HRP deposit was restricted to the superficial layers of the colliculus, anterogradely labeled fibers and retrogradely labeled neurons were observed in the ipsilateral suprageniculate nucleus. However, upon HRP injection extended into the intermediate layers of the colliculus, the number of labeled fibers and neurons was not only increased ipsilaterally but were also observed in the contralateral suprageniculate nucleus. The density of the labeled fibers and the number of labeled neurons was always greater in the ipsilateral side. These results show that the suprageniculate nucleus and the superior colliculus are connected reciprocally and bilaterally, with an ipsilateral dominance.